PROJECT SUMMARY This proposal examines the mechanisms by which G protein-coupled receptors (GPCRs) signal pain. Chronic pain is a hallmark of disease, a side effect of therapy, and a major cause of suffering. Although GPCRs mediate all aspects of nociception and are major therapeutic targets, the mechanisms by which GPCRs signal sustained pain are poorly understood, and clinical trials of GPCR antagonists in chronic pain often fail for unexplained reasons. The proposal challenges three dogmas that contribute to this lack of understanding: 1. GPCRs signal only from the cell-surface. 2. Endosomes are merely a conduit for GPCR recycling or degradation. 3. Cell-surface GPCRs are the optimal therapeutic target. The proposal hypothesizes that: 1. Endosomal GPCRs generate sustained signals that mediate persistent excitation of spinal neurons and nociception. 2. Targeting endosomal rather than cell-surface GPCRs is the ideal therapeutic strategy, and the clinical failure of conventional antagonists relates to their inability to inhibit endosomal receptors. Experiments will focus on substance P and calcitonin gene-related peptide receptors, which mediate central pain transmission and are internalized after painful stimuli. The contribution of receptor endocytosis to nociception will be evaluated using pharmacological and genetic approaches to disrupt clathrin, dynamin and β-arrestin, and by studying transgenic mice expressing non-internalizing receptors. Lipid-conjugation and nanoparticle- encapsulation will be used to deliver antagonists to endosomal GPCRs. Aim 1 will determine the contribution of endocytosis to somatic and colonic nociception in conscious mice. Aim 2 will define the importance of endocytosis for excitation of spinal neurons, which will be analyzed in intact tissues using electrophysiology. Aim 3 will determine the requirement of endocytosis for the generation of signals in subcellular compartments that underlie neuronal excitation and nociception, which will be studied in isolated neurons using biophysical, imaging and proteomic approaches. The results will provide fundamental information about pain signaling and therapy. Since GPCRs are the largest class of signaling proteins and the target of one half of therapeutic drugs, the outcomes will be broadly significant.